The field of this invention is enzyme assays, and in particular, assays involving multiplexed substrates and, optionally multiplexed enzymes.
Blakesley V. A., Scrimgeour A., Esposito D., Roith D. L., Signaling via the insulin-like growth factor-I receptor: does it differ from insulin receptor signaling?, Cytokine and Growth factor reviews (1996), Vol. 1, 2, pp. 153-159.
Bunemann M., Hosey M. M., G-protein coupled receptor kinases as modulators of G-protein signaling, J. of Physiology (1999), 517, 1, pp.5-23.
Cantley, L. C. et al Signal Transduction in Health and Disease, Advances in Second Messenger and Phosphoprotein Research, Vol. 31, 41-48, 1997.
Cohen, C. B. et al Anal. Biochem. Vol 273, 89-97, 1999. Pike L. J., Gallis B., Casnellie J. E., Bornstein P., Krebs E. G., Epidermal growth factor stimulates the phosphorylation of synthetic tyrosine-containing peptides by A431 cell membranes,PNAS, USA 79 (1982), pp.1443-1447.
Nanjee, M. N. Clinical Chemistry, Vol. 42(6), 915-926, 1996.
Osorio C. R. et al Dis. Aquat. Organ. Vol. 40(3), 177-183, 2000.
Pike L. J., Kuenzel E. A., Casnellie J. E., Krebs E. G., A comparison of insulin- and epidermal growth factor-stimulated protein kinases from human placenta, J. of Biol. Chem. (1984), 259, 15, pp.9913-9921.
Sasaki N., Rees-Jones R. W., Zick Y., Nissley S. P., Rechler M. M., Characterization of insulin-like growth factor I-stimulated tyrosine kinase activity associated with the xcex2-subunit of type I insulin-like growth factor receptor of rat liver cells, J. of Biol. Chem. (1985), Vol. 260, 17, pp.9793-9804.
White M. F., Kahn C. R., The Insulin Receptor and Tyrosine Phosphorylation, The Enzymes, Vol. XVII, pp.248-310, 1986.
White, M. F. et al The Enzymes Vol. XVII, 247-311, 1986.
Zhou M., Felder S., Rubinstein M., Hurwitz D. R., Ullrich A., Lax I., Schlessinger J., Real time measurements of kinetics of EGF binding to soluble EGF receptor monomers and dimmers support the dimerization model for receptor activation, Biochemistry (1993), 32, 8193-8198.
There are numerous situations where one is interested in performing a multiplicity of determinations in a single vessel and being able to individually determine the result of each determination. In order to be able to achieve the individual results, it is necessary that each determination be independent of the other determinations, and that each determination provide a product that can be measured and distinguished from the products of the other determinations.
One of the areas of interest is the effect of a change in environment on a plurality of enzymes. In screening compounds for biological activity, one is interested in the effect of the compound on one or more targets, as well as the effect of the compound on enzymes that are not targets. Therefore, if one can perform a single determination under the same conditions, so as to determine the effect of the compound on a plurality of enzymes, one can not only determine the biological activity of the compound as to targets of interest, but also the specificity of the compound in relation to side effects.
Besides screening compounds for biological activity, there is also an interest in determining the effect of a change in environment on cellular activity, as to specific enzymes. For cancer cells, one would be interested in determining changes in the cellular expression of proteins, the activity of individual enzymes, or an enzyme profile in relation to a compound or course of treatment.
One special class of enzymes for which enzyme multiplexing would be advantageous is cell-surface or intracellular receptors, which represent a significant class of targets for drug screening because the receptors are involved in cell growth and metabolism. Many receptors have enzyme domains, such as the insulin receptor, insulin-like growth factor receptor, epidermal growth factor, and platelet-derived growth factor (White). Some receptors can be coupled to enzyme for assaying the receptors (Bunemann). When a ligand binds to the specific site of a receptor, it typically activates the receptor""s enzyme domain. In many cases, the enzyme is protein kinase that can be measured by determination of the rate of phosphorylation of a synthetic peptide. Since many receptors have high affinity to their specific ligands (Pike, 1984, Blakesley, Sasaki), it is possible to develop a method to perform the receptor assay by monitoring their enzyme (kinase) activity. Since only the specifically bound, natural ligand has the potential to activate the enzyme domain, the receptor assay by monitoring its enzyme activity may avoid the non-specific binding issue. This approach can be used for both ligand binding assay and enzyme assay for many receptors, especially for hormones and growth factor receptors.
In screening for compounds that are capable of inhibiting ligand/receptor binding, there are two, and sometimes three, variables that must be screened. The first variable is the test compound itself, e.g., a large number of test compounds derived from a library of potential inhibitors of ligand/receptor interaction. The second variable is test-compound concentration, relative to the concentration of receptor ligand. The third variable is the effect of the test compound on other enzymes, e.g., other receptor kinases on or in the target cell. Testing for and optimizing the concentration of test compounds, and assaying their effect on other cellular enzymes ultimately requires a large number of assay samples. By combining one or more of the test variables in a multiplexed assay, one could substantially reduce the number of assay that needed to be performed and/or provide an internal control for multiple assays.
In one aspect, the invention includes a multiplexed enzyme assay method. The method includes performing a plurality of enzyme reactions in the presence of plurality of enzymes substrates in a set of substrates, under conditions effective to convert an enzyme substrate to a corresponding product, where the product of each substrate and the substrate have different separation characteristics from each other and from the other substrates in the set and their corresponding products. After performing the reactions, the products, and preferably also the substrates in the reactions are separated in a single separation medium. For each separated product and substrate, a separation characteristic effective to identify that product and substrate and a signal related to the amount of the product and substrate is detected. From this, the amount of substrate converted to the corresponding product in each of the reactions can be determined.
The separation characteristic of the substrates and products is preferably electrophoretic mobility in an electrophoretic medium under the influence of an electric field, although other separation characteristics, such as behavior when separated by exclusion or ion-exchange chromatography, isoelectric focusing or mass spectroscopy are also contemplated. The substrates and corresponding products may be fluorescent-labeled, where the detecting includes detecting the fluorescent signal from each product when irradiated with a fluorescence-excitation wavelength.
In one general embodiment, the plurality of enzyme reactions are carried out in a single reaction mixture containing a plurality of different enzymes, where each of the enzymes being assayed is effective to convert one of the substrates in the reaction mixture to the corresponding product. The embodiment may be used, for example, to determine the levels of activity of each of a plurality of different enzymes in a cell, under selected cell conditions, where the different enzymes in the reaction mixture are obtained from the cell under such selected cell conditions.
For use in determining changes in the levels of activity of each of a group of enzymes in a cell, in both control and test cell conditions, the performing, separating, detecting, and determining steps are carried out for enzymes obtained from the cells in both the control and test conditions. For example, to determine changes in the levels of activity of each of a group of enzymes in a cell, when the cell is exposed to an agent known or being tested for its ability to inhibit or activate the level of the activity of one or more of the different enzymes, the performing, separating, detecting, and determining steps are carried out for enzymes obtained from the cells before and after exposure to the agent. Exemplary groups of enzymes include receptor-kinase enzymes and cell-signaling pathway enzymes.
In another general embodiment, for use in assaying the effect of one or more agents in inhibiting or stimulating the activity of a selected enzyme, the enzyme reactions are performed in separate reaction mixtures, where each mixture contains (i) one or more enzymes and, (ii) one or more substrates from the set of substrates.
In a more specific embodiment, for use in screening for or evaluating the ability of test compound to affect enzyme activity, the reaction mixtures also contain one or both of (a) one of a plurality of different test agents and/or one of a plurality of different concentrations of a single agent. The reaction mixtures are combined prior to separating the substrates and products of the different reactions.
The different substrates in a set may include (i) an enzyme substrate moiety, (ii) a mobility modifier that imparts to each substrate and its corresponding product in the set, a unique separation characteristic with respect to the separation characteristics of other substrates and corresponding products in the set, and (iii) a reporter moiety that permits detection of a signal from said substrates and products in the set. Where the substrates in a set have an oligopeptide substrate moiety, and the mobility modifier may be (i) non-peptide moieties coupled to the oligopeptide, (ii) one or more amino acid substitutions in said oligopeptide that preserve the substrate moiety but alter the molecular weight and/or charge of the oligopeptide, and (iii) different reporter moieties with different charges and/or molecular weights.
For use in assaying interactions between a receptor enzyme and a ligand known to stimulate the receptor-enzyme activity, the separate enzyme reaction mixtures preferably include (i) the one or more selected enzymes (ii) one or more substrates from the set of substrates, and (iii) one or more different concentrations of the ligand, including, in at least one reaction mixture, a concentration of ligand sufficient to produce optimal activation of the enzyme. One of the reaction mixtures preferably contains no ligand, to provide an enzyme activity level of non-activated enzyme.
The receptor enzyme may be, for example, a receptor-kinase enzyme effective, in an activated state, to phosphorylate an oligopeptide substrate, such as EGFR, where the ligand is EGF, receptor II kinase, where the ligand is insulin, and insulin receptor kinase, where the ligand is insulin. In this receptor-enzyme assay, the substrates in the set include the oligopeptide substrate, and a mobility modifier of the type noted above.
For assaying the ability of one or more test agents to interfere with ligand-activated enzyme activity, at least some of the reaction mixtures contain, in addition to an optimal concentration of ligand, one of a plurality of different test agents. Alternatively, or in addition, at least some of the reaction mixtures contain, in addition to an optimal concentration of ligand, one or more concentrations of at least one test agent.
In another aspect, the invention includes a set of enzyme substrates for performing a multiplexed enzyme assay. The set includes a plurality of enzyme substrates, each having (i) an enzyme substrate moiety at which an enzyme in the assay reacts with the substrate, to convert it to the corresponding product, (ii) a mobility modifier that imparts to each substrate and its corresponding product in the set, a unique separation characteristic with respect to the separation characteristics of other substrates and corresponding products in the set, and (iii) a reporter moiety that permits detection of a signal from said substrates and products in the set. The reporter moiety may be, for example, a fluorescent reporter. The enzyme may be, for example, a kinase from different functional groups of kinases, including cyclic nucleotide-regulated protein kinases, protein kinase C, kinases regulated by Ca2+/CaM, cyclin-dependent kinases, ERK/MAP kinases, and protein-tyrosine kinases. The kinase may be a protein kinase enzyme in a signaling pathway, effective to phosphorylate an oligopeptide substrate, such as ERK kinase, S6 kinase, IR kinase, P38 kinase, and AbI kinase. In this enzyme assay, the substrates in the set include the oligopeptide substrate, and a mobility modifier of the type noted above. Other kinases of interest may include, for example, Src kinase, JNK, MAP kinase, cyclin-dependent kinases, P53 kinases, platelet-derived growth factor receptor, epidermal growth factor receptor, and MEK.
For use in a method for assaying one or more enzymes having an oligopeptide substrate, the substrates in a set have an oligopeptide substrate moiety, and the mobility modifier may be (i) non-peptide moieties coupled to the oligopeptide, (ii) one or more amino acid substitutions in the oligopeptide that preserve said substrate moiety but alter the molecular weight and/or charge of the oligopeptide, and (iii) different reporter moieties with different charges and/or molecular weights.
These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.